Modification and clearance of low density lipoproteins during the formation of endotoxin-lipoprotein complexes

Changes in electrical charge and clearance rate of LDL after the formation of their complexes with bacterial LPS were studied in experiments on Wistar rats. It was found that binding of S. minnesota R595 LPS with ¹²⁵I-LDL sharply accelerated clearance of the greater part of LDL complexes, but on the other hand induced the appearance of an LDL-LPS subfraction with slower elimination rate compared to free LDL. Electrophoresis showed that after binding of LPS, LDL acquired a negative charge. These data suggest that the formation of LDL-LPS complexes is accompanied by modification of LDL due to which they acquire atherogenic properties. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 145, No. 4, pp. 408–410, April, 2008     

Evaluation of monoclonal antibodies to human plasma low density lipoproteins. A requirement for lipids to maintain antigenic structure

Human plasma low density lipoproteins (LDL) are composed of approximately 25% apoproteins and 75% lipids (w/w). Immunochemical properties of LDL were studied using monoclonal antibodies. BALB/c mice were immunized with LDL and the spleen cells from these mice were then fused with a non-immunoglobulin secreting myeloma cell line (F0). The clones producing desirable antibodies were selected to study the antigenic properties of LDL by enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay. First, it was found that the maximal binding of 125I-labeled LDL to polyvinyl chloride microtiter dishes was not temperature dependent. The binding affinity was high with a Ka value of approximately 1.9 X 10(10) M-1 while the monoclonal antibodies possessed an affinity to LDL of 5 X 10(8) M-1 which was 2 orders less than the affinity of LDL to the dishes. The former binding, once established, was irreversible as judged by a subsequent incubation with an excess of unlabeled LDL. The latter binding could be displaced by unlabeled LDL. Therefore, the ELISA technique offered a satisfactory approach to study the interaction between LDL and monoclonal antibodies. Removal of lipids from bound LDL by organic extraction resulted in a 50% loss of immunoreactivity, suggesting that the lipids of LDL are important in maintaining the antigenic structure of LDL. Since the apoprotein of LDL also constitutes approximately 40% of the mass (w/w) of very low density lipoproteins (VLDL), the immunoreactivity of VLDL assessed by LDL-monoclonal antibodies was also carried out. Removal of triglycerides from VLDL by lipoprotein lipase resulted in a substantial loss of immunoreactivity as determined by radioimmunoassay. These findings are consistent with the concept that lipids play a role in maintaining the integrity of the antigenic structure of LDL.     

内源性高甘油三酯血症患者血浆VLDL、LDL及HDL对血凝的影响

目的：研究内源性高甘油三酯血症(HTG)患血浆极低密度脂蛋白(VLDL)、低密度脂蛋白(LDL)及高密度脂蛋白(HDL)是否发生了氧化修饰及其对血凝的影响。方法：对2l例内源性高甘油三酯血症患与2l例年龄性别相近的正常人的血脂、脂质过氧化物进行了分析。用一次性密度梯度超速离心法分离血浆VLDL、LDL及HDL,测定这三种脂蛋白的234nm光吸收、相对电泳迁移率(REM)和硫代巴比妥酸反应物质(TBARS),分别将这三种脂蛋白加入由正常人新鲜混合血浆构成的反应系统中,按试剂盒分别测定凝血酶原时间(PT)及活化部分凝血酶原时间(APIT)。结果：内源性HTG患血浆TG含量平均升高2．73倍,HDLC下降l.7l倍,同时LPO升高1．22倍;HTG组VLDL、LDL及HDL的REM、234nm光吸收值、TBARS含量均较对照组显增加(P＜0．01),表明内源性HTG患血浆VLDL、LDL及LDL均发生了氧化修饰生成Ox—VLDL、Ox-LDL.PT及APTT在分别加入HTG组的VLDL、LDL及HDL后均比加入相应正常组脂蛋白明显缩短(P均＜0．05)。相关分析表明,HTG组血浆VLDL及HDL相对电泳迁移率(REM)与PT呈负相关(P＜0．01)。结论：HTG患血浆VLDL、LDL及HDL发生了氧化修饰,并使PT及APTT明显缩短。     

Production of mouse inflammatory macrophage hybridomas

Summary A procedure for preparing hybridomas from murine inflammatory macrophages is described. The resulting hybrids express Ia antigens on their surfaces and exhibit various degrees of bactericidal, phagocytic, and tumoricidal activities. When irradiated, they are very useful for phagocytosing cellular debris in other cell cultures and can temporarily replace the need for spleen irradiated filler cells in T-cell cultures. The macrophage hybridomas seem to remain stable in long-term culture, although some functional properties have been noted to change. In this manner, relatively homogeneous populations of macrophages can be obtained for study. Their functional properties may vary depending on the fusion partner and the source of macrophages.     

Binding of human lipoproteins (low, very low, high density lipoproteins) to recombinant envelope proteins of hepatitis C virus

Heterogeneities in the density of hepatitis C virus (HCV)-RNA-carrying material from human sera (1.03–1.20 g/ml) are partially due to the binding of lipoproteins [low density (LDL), very low density (VLDL), high density (HDL) lipoproteins] and immunoglobulins. In this study we demonstrate the binding of recombinant HCV envelope protein (E1/E2) to human LDL, VLDL and HDL on a molecular basis. The binding of lipoproteins was restricted to the middle part of the E1 gene product (amino acids 222–336) and the C-terminal part of the E2 protein (amino acids 523–809). Lipoproteins did not bind to recombinant HCV core protein. Received: 22 December 1999     

氧化低密度脂蛋白通过氧化应激诱导小鼠造血干细胞衰老

 目的 探讨氧化低密度脂蛋白（ox-LDL）体外诱导造血干细胞(HSCs)衰老的可能机制。方法 用免疫磁性分选法分离纯化小鼠HSC,与ox-LDL共培养,采用β-半乳糖苷酶（SA-β-Gal）染色检测衰老HSC,流式细胞术检测HSC细胞周期分布,混合集落培养(CFU-Mix)检测HSC混合集落形成能力。流式细胞术和免疫荧光检测HSC产生活性氧（ROS）的量,酶学比色法检测HSC培养上清液超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）及丙二醛（MDA）含量。Southern blot和TRAP-PCR法检测HSC端粒长度和端粒酶活性。结果 ox-LDL诱导HSC呈现典型的衰老生物学表现：SA-β-Gal染色阳性细胞率显著增高（P <0.01）; G0/G1 期比例明显增加,S 期显著减少（P <0.01） ;CFU-Mix数量显著减少（P <0.01）。衰老HSC端粒缩短（P <0.05）,端粒酶活性降低（P <0.05）。衰老HSC ROS含量显著增加（P <0.01）,细胞培养上清液中SOD、GSH-Px活力下降、 MDA含量增加（P <0.05）。 结论 ox-LDL能通过氧化应激诱导HSC衰老,其机制可能与ROS的蓄积及抗氧化酶活性受抑引起端粒功能异常有关。     

Genetics of the low density lipoprotein receptor:

Fibroblast low density lipoprotein (LDL) plasma membrane receptor activity, measured as 125I-LDL association (plasma membrane binding plus intracellular accumulation) and degradation was determined in cell strains from 14 monozygotic (MZ) and 21 like-sexed dizygotic (DZ) normolipidemic twin pairs. The twins were between 57 and 62 years old and had liver apart for an average of 38 years (range 0-60). The intrapair differences were significantly smaller in MZ than in DZ twin pairs in fibroblast 125I-LDL association as well as degradation assays (P less than 0.05). These findings suggest a genetic influence on normal variation in LDL receptor activity in vitro. In two MZ pairs discordant for psoriasis, the psoriatic twin had markedly lower LDL receptor activity than the cotwin.     

Protein-bound lipids in human low density lipoproteins

In vitro oxidation of low density lipoproteins is found to be accompanied by accumulation of sterol and phospholipid residues covalently bound with apolipoprotein B. The content of protein-bound lipids in the subfraction of desialylated low density lipoproteins from healthy subjects and patients with coronary atherosclerosis is shown to be higher than that in native lipoproteins. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 120, No. 8, pp. 155–157, August, 1995 Presented by E. I. Chazov, Member of the Russian Academy of Medical Sciences     

Fusion of mouse-mouse cells to produce hybridomas secreting monoclonal antibody

Summary A procedure is described for fusing and producing large numbers of hybridomas using mouse myeloma cells and immunized mouse spleen cells. The medium used is a modified Dulbeceo's minimum essential medium, supplemented with a low percentage of serum.     

Lp(a) lipoprotein enters cultured fibroblasts independently of the plasma membrane low density lipoprotein receptor

Lp(a) lipoprotein shares the apoB antigen with low density lipoprotein (LDL). The Lp(a) antigen is unique for Lp(a) lipoprotein. Fibroblast association (i.e. plasma membrane binding plus intracellular accumulation), plasma membrane binding, intracellular accumulation and degradation of 125I-Lp(a) lipoprotein were studied in strains from subjects with or without autosomal dominant hypercholesterolemia (HC). Subjects without HC (non-HCs) have cell surface receptors for low density lipoprotein (LDL receptors). On the average, HC heterozygotes have half-normal LDL receptor activity and "receptor-negative" HC homozygous cell strains lack functional receptors. Fibroblast processing of 125I-Lp(a) lipoprotein was compared to fibroblast processing of 125I-LDL. LDL receptor-dependent processing of 125I-LDL was saturated at about 50 microgram apo 125I-LDL.ml-1 in non-HC fibroblasts. 125I-Lp(a) lipoprotein was, however, largely processed independently of receptor mechanisms by non-HC cells (highest concentration examined 150 microgram apo 125I-Lp(a) lipoprotein . ml-1). Lp(a) lipoprotein did not interfere with 125I-LDL for fibroblast association, but inhibited 125I-LDL degradation. The interference with 125I-LDL degradation was time dependent. Only slightly higher 125I-Lp(a) lipoprotein processing values were found in non-HC and HC heterozygous strains than in "receptor-negative" HC homozygous strains. However, non-HC cells had more than tenfold higher 125I-LDL processing values than "receptor-negative" HC homozygous cells.     

The adaptation of the plaque reduction assay for measuring specific cytotoxic cells in limiting dilution cultures

The use of the original haemolytic plaque reduction technique to measure cytotoxic T lymphocytes (CTL) has been developed further as a rapid screening assay, particularly suitable for limiting dilution analyses. Using hybridoma cells as targets, the cytotoxicity has been measured by the loss of haemolytic plaque formation and by the reduction of the amount of haemolytic monoclonal antibody secreted from viable target cells into the assay supernatants. The assessment of large numbers of cytotoxic samples has been greatly facilitated by quantitating the amount of haemoglobin released in the assay with an automated microELISA multiscanner and by scoring visually using a modification of the spot test. Using these new techniques, relatively high frequency estimates of cytotoxic cell precursors in an allogeneic response (1 in 462 spleen cells) and an anti-fluorescein response (1 in 3970 spleen cells) were obtained.     

Characterization of low density lipoprotein receptor (LDLR) gene mutations in Albania

Introduction

Familial hypercholesterolaemia (FH) is a clinical syndrome characterised by elevated serum total cholesterol (TCHOL) levels due to an increase in low-density lipoprotein (LDL) cholesterol, by tendon xanthomata and clinical manifestations of ischaemic heart disease in early life. Typically, it results from mutations in the low-density lipoprotein receptor (LDLR) gene. So far, more than 800 mutations have been reported for the LDLR gene and account for FH. The nature of LDLR gene mutations varies among different ethnicities. Until now no mutations of LDLR have been reported in the Albanian population.

Material and methods

We assessed the contribution of the LDLR gene mutations as causes of FH in an Albanian population. Fifty probands with a clinical diagnosis of FH were included. We analysed all the exons and the promoter of the LDLR gene by using restriction isotyping or direct sequencing.

Results

Twenty-one patients were heterozygous for the 1646G>A mutation (FH Genoa) in exon 11 and 9 patients were heterozygous for the 81T>C mutation in exon 2 of the LDLR gene.

Conclusions

This report describes two LDLR gene mutations accounting for FH in Albania (1646G>A, 81T>C).     

Cloning of lymphocytes from whole blood by limiting dilution

A simple whole blood limiting dilution technique was developed to clone human lymphocytes non-specifically. The geometric mean frequency of clone forming cells in 13 normal individuals was found to be 31.1%. Compared with measurement of proliferation in mass suspension culture, cloning provides a quantitative and easily interpretable endpoint for a variety of lymphocyte studies and its use in measurement of radiation sensitivity is presented as an example.     

氧化低密度脂蛋白下调血管内皮细胞表达Rictor

目的探讨氧化低密度脂蛋白对培养血管内皮细胞表达基因Rictor的影响。方法分离并培养人脐静脉内皮细胞,用不同浓度(10、20、40和80 mg/L)氧化低密度脂蛋白作用24 h后,用RT-PCR及Western blot检测Rictor的表达情况,并检测转染Rictor后,蛋白激酶Akt及eNOS磷酸化的变化,用硝酸还原酶还原法测定NO释放量。结果氧化低密度脂蛋白显著降低Rictor的mRNA及蛋白的表达量(P<0.01),使Rictor-mTOR复合物形成减少。转染Rictor后,不仅Rictor表达增加而且使蛋白激酶Akt及eNOS磷酸化增加;内皮细胞NO释放量增多(P<0.05)。结论影响内皮细胞表达Rictor,是氧化低密度脂蛋白诱发血管内皮细胞功能不良的重要机制之一。     

氧化低密度脂蛋白和一氧化氮合酶在人主动脉内膜中的表达及相关意义

目的：研究氧化低密度脂蛋白(OX-LDL)和一氧化氮合酶(NOS)阳性细胞在正常成人及动脉粥样硬化患者主动脉内膜中的表达及相关意义。方法：选取正常成人和动脉粥样硬化患者主动脉标本,以免疫组化SP法、NADPH—d酶组化方法分别检测OX—LDL和NOS阳性细胞在内膜中的表达。结果：动脉粥样硬化的主动脉壁内膜层OX—LDL阳性表达强于正常的主动脉,而NOS阳性表达弱于正常的主动脉,且两种表达呈负相关。结论：主动脉壁内膜沉积的OX—LDL可抑制内膜细胞NOS活性,OX—LDL和NO在动脉粥样硬化过程中有相互促进的共同致病机制。     

Genetics of the low density lipoprotein receptor:

Fibroblast association (plasma membrane binding plus intracellular accumulation) and degradation of radioiodinated low density lipoprotein (125I-LDL) index plasma membrane LDL receptor activity. Cultured fibroblasts from 23 subjects affected with familial hypercholesterolemia (HC) and from 95 subjects without HC (non-HCs) were tested for 125I-LDL association and degradation. Both LDL receptor activity indices were twice as high in non-HC and HC heterozygous cell strains. This is compatible with a major gene effect on LDL receptor activity. However, a considerable overlap between non-HC and HC heterozygous values was found in the 125I-LDL association assay [median (range) 970 (330-2500), and 450 (250-490), respectively] and in the degradation assay [median (range) 810 (280-2020), and 470 (160-790), respectively]. The values are expressed as ng 125I-LDL X mg cell protein-1 X 4.5 h-1. These great overlaps in the LDL receptor activity indices support the view that the influence of LDL receptor activity on the HC phenotype may be smaller than believed previously. Furthermore, for the diagnosis of HC, these LDL receptor activity assays are far more expensive and have less sensitivity and specificity than simple serum cholesterol determination. The LDL receptor-dependent 125I-LDL association values for the HC heterozygous individuals clustered into four groups. Family data supported the hypothesis that this variation could be due to four different LDL receptor variants, each coded for by different alleles at the LDL receptor locus. If confirmed, this finding may have implications for the understanding of the variable expression of HC and also of the genetic impact on lipoprotein metabolism and susceptibility to atherosclerosis in non-HCs.     

The influence of murine macrophage-conditioned medium on cloning efficiency, antibody synthesis, and growth rate of hybridomas

Murine B-cell hybridomas made with the P3X63-AG8.653 myeloma showed increases in cloning efficiency and efficiency of growth in hypoxanthine-aminopterin-thymidine (HAT) medium of 50-100-fold in the presence of medium conditioned by primary mouse peritoneal macrophages (MCM). Similar effects were elicited by MCM from 3 continuous macrophage lines. The J774A.1 line conditioned the medium as efficiently as primary macrophages without induction. Conditioning by the P388D1 line was several-fold less efficient, but could be increased by treating the cells with Escherichia coli lipopolysaccharide. By contrast, the BJ-1 macrophage line required treatment with the lipopolysaccharide to induce expression of the hybridoma growth factor(s). Four commercially available serum supplements could not substitute for MCM, but addition of MCM and the supplements together stimulated the growth rate of hybridomas in media with 4% or less fetal bovine serum. The rate of antibody synthesis paralleled the growth rate, and the amount of antibody synthesized per cell was approximately the same for hybridomas grown in medium supplemented with MCM or adapted to growth in the absence of MCM. The results indicate that MCM has advantages as an alternative to 'feeder cells' and serum supplements in hybridoma cultures, and suggest that MCM may be useful for hybridoma culture at reduced serum concentrations. The nature of the soluble factor(s) in MCM which promote these effects remains unknown.     

A 9.6 kilobase deletion in the low density lipoprotein receptor gene in Norwegian familial hypercholesterolemia subjects

Haplotype analysis of the low density lipoprotein receptor (LDLR) gene was performed in Norwegian subjects heterozygous for familial hypercholesterolemia (FH). Southern blot analysis of genomic DNA, using an exon 18 specific probe and the restriction enzyme NcoI, showed that two out of 57 unrelated FH subjects had an abnormal 3.6 kb band. Further analyses revealed that this abnormal band was due to a 9.6 kb deletion that included exons 16 and 17. The 5' deletion breakpoint was after 245 bp of intron 15, and the 3' deletion breakpoint was in exon 18 after nucleotide 3390 of cDNA. Thus, both the membrane-spanning and cytoplasmatic domains of the receptor had been deleted. A polymerase chain reaction (PCR) method was developed to identify this deletion among other Norwegian FH subjects. As a result of this screening one additional subject was found out of 124 subjects screened. Thus, three out of 181 (1.7%) unrelated Norwegian FH subject possessed this deletion. The deletion was found on the same haplotype in the three unrelated subjects, suggesting a common mutagenic event. The deletion is identical to a deletion (FH-Helsinki) that is very common among Finnish FH subjects. However, it is not yet known whether the mutations evolved separately in the two countries.     

家族性高胆固醇血症家系低密度脂蛋白受体基因剪接突变的研究

目的　检测中国汉族家族性高胆固醇血症 (familial hypercholesterolemia,FH)大家系低密度脂蛋白受体 (low density lipoprotein receptor,L DL R)基因突变 ,探讨 FH发病的分子机理。方法　首先采用聚合酶链反应 -限制性片段长度多态性 (polymerase chain reaction- restriction fragment lengthpolymorphism,PCR- RFL P)技术检测载脂蛋白 B1 0 0 (apo B1 0 0 )基因 Q35 0 0 R突变 ,排除家族性 apo B1 0 0 缺陷症 ,再采用 PCR扩增结合核苷酸序列分析检测 1例临床诊断为 FH纯合子患儿及其家系成员 L DL R基因启动子和全部 18个外显子片段 ,结果与 Gen Bank公布的该基因正常序列对比找出突变 ,并在家系其他成员中证实该突变。结果　该患儿 L DL R基因第 3内含子剪接供体处存在 IN 5′GT→AT纯合剪接突变 ,并且在家系中得到证实 ,一级和二级亲属中各发现 2例相同位点和相同形式的杂合子 ,其基因型表现为野生型和突变型杂合现象。同时未检测出患儿及其父母 apo B1 0 0 Q35 0 0 R突变。结论　发现 L DL R基因第 3内含子 G→ A纯合剪接突变 ,可能是该 FH家系发病的分子基础 ;检测该突变对临床干预和遗传指导有参考价值。